IJC

International Journal of Cancer

Messenger RNA vaccine based on recombinant MS2 virus-like

particles against prostate cancer
Jinming Li1, Yanli Sun1,2, Tingting Jia1,2, Rui Zhang1, Kuo Zhang1 and Lunan Wang1
1
National Center for Clinical Laboratory, Beijing Hospital of the Ministry of Health, Beijing 100730, People’s Republic of China
2
Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, People’s Republic of China

Prostate cancer (PCa) is the most diagnosed cancer in the western male population with high mortality. Recently, alternative
approaches based on immunotherapy including mRNA vaccines for PCa have shown therapeutic promise. However, for mRNA
vaccine, several disadvantages such as the instability of mRNA, the high cost of gold particles, the limited production scale
for mRNA-transfected dendritic cells in vitro, limit their development. Herein, recombinant bacteriophage MS2 virus-like par-
ticles (VLPs), which based on the interaction of a 19-nucleotide RNA aptamer and the coat protein of bacteriophage MS2, suc-
cessfully addressed these questions, in which target mRNA was packaged by MS2 capsid. MS2 VLP-based mRNA vaccines
were easily prepared by recombinant protein technology, nontoxic and RNase-resistant. We show the packaged mRNA was
translated into protein as early as 12 hr after phagocytosed by macrophages. Moreover, MS2 VLP-based mRNA vaccines
induced strong humoral and cellular immune responses, especially antigen-specific cytotoxic T-lymphocyte (CTL) and balanced
Th1/Th2 responses without upregulation of CD41 regulatory T cells, and protected C57BL/6 mice against PCa completely. As
a therapeutic vaccine, MS2 VLP-based mRNA vaccines delayed tumor growth. Our results provide proof of concept on the effi-
cacy and safety of MS2 VLP-based mRNA vaccine, which provides a new delivery approach for mRNA vaccine and implies
important clinical value for the prevention and therapy of PCa.

The incidence rates of prostate cancer (PCa) remain elevated immune system to fight PCa, including monoclonal antibod-
in the developed countries worldwide including North Amer- ies (e.g., Ipilimumab, augmenting immune responses to

Tumor Immunology
ica, Oceania and western and northern Europe, and the mor- tumors by blockade of cytotoxic T-lymphocyte (CTL) antigen
tality rates tend to be higher in less developed regions of the 42, dendritic cells (DCs) vaccines (e.g., Sipuleucel-T, present-
world.1 Over the past decades, several therapeutic approaches ing the epitopes of prostate acid phosphatase (PAP) to CTLs
for PCa including chemotherapy, radiotherapy, surgery and through activated DCs3, viral vector vaccine (e.g., Prostvac, a
immunotherapy provide survival benefits. The most attractive prostate-specific-antigen-directed vaccine4, DNA vaccines5,6
approach is immunotherapy that modulate the patient’s and RNA vaccine (e.g., CureVac, a free and protamine-
complexed mRNA vaccine7. Among these modalities, mRNA
Key words: RNA vaccine, MS2 bacteriophage, virus-like particles, vaccine is considered as an efficient alternative to protein and
prostate cancer, cytotoxic T cell DNA vaccines not only because of its antigenic potential and
Abbreviations: DC: dendritic cell; GM-CSF: granulocyte-macro- immunostimulating property8 but also because mRNA is
phage colony-stimulating factor; PCa: prostate cancer; RT: reverse safe, low cost, highly pure and prepared easily.9
transcription; TEM: transmission electron microscopy; VLP: virus- Despite protein expression following the injection of
like particle mRNA in vivo was observed as early as 1990,10 the instability
Additional Supporting Information may be found in the online of naked RNA vaccines limited its application.11 Several
version of this article. approaches has been used to improve intracellular stability
Grant sponsor: National Natural Science Foundation of China; and translational efficacy of mRNA, including liposome-
Grant number: 81171981; Grant sponsor: National High entrapped mRNA,12 replicative mRNA,13 mRNA-coated gold
Technology Research and Development Program of China; Grant particles14 and in vitro mRNA-transfected DCs.3,15 All these
number: 2011AA02A116 approaches require a large amount of mRNA produced
DOI: 10.1002/ijc.28482 through the utilization of enzymes and a plasmid DNA tem-
History: Received 27 June 2013; Accepted 29 Aug 2013; Online 17 plate.16 Furthermore, for all of these approaches, one of the
Sep 2013 greatest challenges is the relative instability of mRNA, which
Correspondence to: Jinming Li, National Center for Clinical results from degradation by prevalent RNase; thus, the neces-
Laboratory, Beijing Hospital of the Ministry of Health, No. 1 Dahua sary steps in generating the mRNA vaccine, including synthe-
Road, Dongdan, Beijing 100730, People’s Republic of China, Tel.: sis, purification, storage and inoculation of mRNA, may
186-10-58115061, Fax: 86-10-65212064, result in ultra-low in vivo concentrations that may elicit a
E-mail:[email protected] weaker immune response.

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What’s new?
Immunotherapy based on mRNA vaccination is a promising means of fighting prostate cancer, but mRNA instability and other
factors could limit its utility. Some of those factors, however, may be overcome with an mRNA vaccine based on bacteriophage
MS2 virus-like particles (VLPs), according to this study. MS2 VLP-based vaccines were prepared using recombinant protein
technology, and when tested in a mouse model were found to be nontoxic and to stimulate humoral and cellular immune
responses, fully protecting mice against prostate cancer.

Besides, the high cost of gold particles and the gene gun Material and Methods
with inconvenient experiment animal bombardment hinders Cells and mice
the development of ballistic delivery of mRNA for vaccina- Prostate cell line TRAMP-C2 was cultured as mentioned else-
tion. In vitro mRNA-transfected DCs have been used for can- where.30 Mouse macrophage cell line RAW264.7 was cultured
cer therapy in clinics, but the in vitro derivation of DCs in in RPMI 1640 medium with 10% fetal bovine serum (FBS)
GMP conditions is costly16 and the source of autologous DCs (Gibco). Both cell lines were obtained from ATCC.
is limited. All of these disadvantages obstruct the application Six to eight-week-old C57BL/6 male mice (the Animal
and development of mRNA vaccines, so there is a high Center of the Academy of Military Medical Sciences, Beijing,
demand to develop a simple, safe, efficient, stable and low- China) were kept under SPF isolation conditions and fed a
cost approach for the delivery of mRNA vaccines. standard diet at the animal facilities of the Tumor Hospital,
Bacteriophage MS2 virus-like particles (VLPs) may be Beijing, China. All animal experiments were approved by the
promising to meet the demand above. On one hand, these Ethics Committee of the National Center for Clinical
particles could be prepared easily by recombinant protein Laboratories.
technology.17 On the other hand, the MS2 capsid, which
interacts with a specific 19-nucleotide stem-loop (pac site),
Construction of plasmids
can package the target RNA located at the 50 terminus of the
The target genes were amplified by PCR using the corre-
pac site, and this protects target RNA from degradation by
sponding primers shown in Supporting Information Figure
nucleases.17,18 To date, MS2 VLP-based RNA have been used
S1 and Table S1, and the corresponding vectors pMS2,
as multi-purpose internal controls for diagnostic purposes,19
pMS2P, pMS2-GFP, pMS2-GM-CSF, pMS2-mPAP, pMS2-
for drug delivery,20,21 or as HIV mRNA vaccine,22 however,
Tumor Immunology

hPAP, pMS2-mPAP-GM-CSF and pMS2-hPAP-GM-CSF

whether this approach can be used for the delivery of mRNA
were constructed as shown in Supporting Information Figure
vaccines for PCa has not yet been investigated.
S1. There is a Gly-Ser linker sequence between the 30 termi-
PAP, as a prostate-extensive antigen, induces specific cellu-
nus of PAP gene and the 50 terminus of mouse GM-CSF.
lar immune response and tumor regression in PCa, whether in
Correct construction of all plasmids was confirmed by
the form of protein,23 peptides24 or DNA.6,25 As was reported,
sequencing.
xenogeneic PAP protein showed stronger immunogenicity
than self-PAP,26 whose homology was approximately 80% at
the amino acid level. However, whether xenogeneic PAP Preparation of MS2 VLP-based mRNA vaccines
mRNA can break self-tolerance and promote antitumor activ- The plasmids pMS2P, pMS2-GM-CSF, pMS2-hPAP, pMS2-
ity has not been reported. Therefore, xenogeneic PAP mRNA mPAP, pMS2-GFP, pMS2-hPAP-GM-CSF and pMS2-mPAP-
as the target antigen for PCa vaccine is investigated here. GM-CSF were transferred into the YPH499 yeast strain (Stra-
The superior adjuvant properties of the granulocyte- tagene, Santa Clara, CA) by the LiAC/ssDNA/PEG method,
macrophage colony-stimulating factor (GM-CSF) protein respectively.31 The expression of MS2 VLP-based mRNA was
were first demonstrated in a gene-modified tumor vaccine performed according to the instruction of pESC yeast epitope
model,27 and was applied in PCa vaccine development, e.g., tagging vectors (Stratagene). When cultures were grown to
Sipuleucel-T, which can activate DCs and present antigen to an OD600 of 8.0, cells were harvested by centrifugation, resus-
both the B-cell and T-cell. In addition, an allogeneic, whole- pended in a final volume of 30 mL TBS (10 mM Tris–HCl,
cell, GM-CSF-secreting cellular immunotherapy for PCa– 100 mM NaCl, 1 mM MgCl2, 0.1 mM EDTA, pH 7.4) and
GVAX combined with Iplimumab prolonged overall sur- disrupted by sonication (Branson Sonifier 350) on ice for 20
vival.28 Murine GM-CSF mRNA co-immunized with the min (on for 5 sec, off for 5 sec). The homogenate was centri-
mRNA encoding tumor-associated antigens has potentiated fuged at 4 C for 30 min at 12,000 rpm. The supernatant was
CTL response.29 collected and MS2 VLP-based mRNA vaccines were recov-
Therefore, in this study, we have constructed a RNase- ered from the supernatant by PEG 6,000/NaCl (20%/1M)
resistant, MS2 VLP-based PAP-GM-CSF mRNA vaccine precipitation. The precipitation was resuspended in 15 mL
against PCa, and evaluated their immunogenicity and antitu- TBS, and then 7.5 mL chloroform was added and mixed with
mor efficacy in mouse model. the solution. The mixture was centrifuged at 4 C for 15 min

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at 12,000 rpm. The supernatant was collected, then the nano- mRNA vaccination
particles were verified by 1% agarose gel electrophoresis with Six to eight-week-old male C57BL/6 mice were intravenously
ethidium bromide staining before and after incubated with vaccinated with 50 lg of total protein of purified VLPs (16.7
DNase I (10 U/mL) and RNase A (10 mg/mL) (Sigma, St 3 1012 RNA copies) resuspended in sterile PBS. The mice
Louis, MO) for 1 hr, and purified by Sephacryl S-200 gel received two boosts with the same amount of VLPs at a fort-
exclusion chromatography (BioLogic DuoFlow chromatogra- nightly interval. Mice injected with PBS alone were used as
phy system). The products were analyzed by SDS–PAGE on controls. Sera were collected before every immunization, and
12.5% gels and were also observed by transmission electron the 14th and 30th days after the last immunization. The
microscopy (TEM) at 75 kV and 200,0003 screen experiments were repeated twice.
magnification.
Serum antibody ELISA
Identification of the mRNA packaged by MS2 capsid using Antibodies reactive with the hPAP (Abcam, Cambridge, UK)
RT-PCR or mPAP (prepared by our laboratory) protein were assayed
RNA isolation and reverse transcription (RT)-PCR reaction by ELISA (see Additional Supporting Information).
were performed as described elsewhere,17 with the appropri-
ate primers listed in Supporting Information Figure S1 and T cell proliferation assay
Table S1. For identification of the RNA packaged by MS2 T cell proliferation assay was performed by EdU incorpora-
capsids, RT-PCR was performed with the corresponding pri- tion32 (see Additional Supporting Information).
mers at 94 C for 5 min, followed by 30 cycles of 30 sec at
95 C, 30 sec at 60 C and 45 sec 1 min 50 sec at 72 C, and
Cytokine ELISA
followed by 5 min at 72 C. PCR products were verified by
Splenocytes from vaccinated mice were isolated 7 days after
1% agarose gel electrophoresis with ethidium bromide stain-
the last vaccination, and were stimulated with 2 lg/mL PAP.
ing, then purified and ligated into the pMD18-T plasmid
Supernatants were collected at 72 hr, and were subjected to
(Takara, China) for further verification by sequencing.
cytokine (IFN-g, TNF-a, IL-12, IL-6 and IL-4) ELISA assay
(see Additional Supporting Information). In addition, serum
Stability assay of MS2 VLP-based mRNA vaccines
levels of IL-12 and TNF-a in C57BL/6 mice were also
Purified MS2 VLP-based mRNA vaccines were incubated
detected 4, 24 and 48 hr after intravenous injection of 50 lg
with 10 U/mL DNase I and 10 mg/mL RNase A for 2, 4, 6, 8,
of MS2 VLPs, hPAP-GM-CSF VLPs or PBS.

Tumor Immunology
10, 12, 14, 16, 18 hr and were fractionated in a 1% agarose
gel with ethidium bromide staining.
IFN-c ELISPOT assay
Murine IFN-g ELISPOT assay were performed 6 days after in
Phagocytosis assay
vitro restimulation (see Additional Supporting Information).
The hPAP-GM-CSF VLPs were fluorescently labeled with FITC
(Sigma), according to the manufacturer’s instructions. 1 3 106
RAW264.7 cells were cultured in 24-well plates, and FITC- FACS analysis of dendritic cells maturation in peripheral
labeled gag VLPs were added to each well with a final concen- blood
tration 10 lg/mL. Unlabeled hPAP-GM-CSF VLPs were used DC maturation in peripheral blood was determined by a sur-
as negative control. After 12 hr incubation, the cells were face marker staining assay with direct FACS analysis on the
washed three times with phosphate buffered saline (PBS) to 7th day after the final immunization (see Additional Support-
remove the hPAP-GM-CSF VLPs-containing medium. Uptake ing Information).
and intracellular distribution of the complex were monitored
with a fluorescence microscope with appropriate filters. Staining of CD41CD251Foxp31 T (Treg) cells in peripheral
blood
Expression of packaged exogenous mRNA in mammalian Treg cells in peripheral blood were analyzed by FACS 7 days
cell line after the final immunization (see Additional Supporting
The GFP VLPs (10 lg/mL) were added to 3 3 105 Information).
RAW264.7 cells in 24-well plates in triplicate, and the expres-
sion of GFP protein was monitored at 0, 24, 48, 72 hr by the Tumor challenge
fluorescence microscope. Additionally, the cells were stained The C57BL/6 mice of the prophylactic groups were injected
with DAPI and red fluorescent reactive dye (Life technology), subcutaneously with 1 3 106 TRAMP-C2 cells in 100 lL
and confocal laser scanning (LSM510 laser scanning micro- PBS 14 days after the last vaccination. The C57BL/6 mice of
scope, Carl Zeiss, Germany) was performed at 72 hr. The the therapeutic groups were first injected subcutaneously with
cells were collected at 24, 48, 72, 96, 120 hr and the RNA 5 3 105 TRAMP-C2 cells in 100 lL PBS, then were vacci-
was extracted from the same number of cells, and analyzed nated with 50 lg of total protein of purified VLPs resus-
by RT-PCR. pended in sterile PBS on days 3, 10, 17 and 24. The

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 1686 The packaged RNA vaccine for prostate cancer

experiments were repeated twice. Tumor sizes of the mice

were measured in three dimensions using calipers once a
week, and survival time and survival rates were monitored.

Statistical analysis
Statistical analyses were performed using GraphPad Prism
Software (Version 5.01). Survival curves were compared with
log-rank test. Grouped data (IgG antibody, tumor growth
kinetics) were performed using two-way analysis of variance
(ANOVA) and Bonferroni post test. Others were compared
using one-way ANOVA test. Data were considered statisti-
cally significant when p < 0.05.

Results
MS2 VLP-based mRNAs with one C-variant pac site are
produced at high yields in yeast
Because the conformation of target protein can be changed
by inserting one pac site carrying two initiation codons
before the initiation codon of the target gene, we cloned only
one pac site directly after the stop codon of the target gene.
To ensure that the encapsidated mRNAs could be translated
in a eukaryotic environment, MS2 VLP-based mRNAs were
expressed in Saccharomyces cerevisiae. The results of agarose
gel electrophoresis with ethidium bromide staining showed
that after MS2 VLP-based mRNA were incubated with
DNase I and RNase A, a band of approximately 1,000 bp in
size remained in every lane (Fig. 1a), indicating that these
mRNAs separated from the ultrasonic supernatant of S. cere-
Tumor Immunology

visiae were nuclease-resistant. The purified MS2 VLP-based

mRNA prepared from 1 L of culture with an OD600 of 8.0
were 2–3 mg/L (Fig. 1b), and the yield was negatively corre-
lated with the length of inserted target genes (Supporting Figure 1. Identification of MS2 VLP-based mRNAs. (a) Nuclease-
Information Fig. S2). Furthermore, SDS-PAGE results resistance assay of VLPs. The VLPs in Lanes 2, 4, 6, 8, 10 and 12 were
revealed that the molecular weight for MS2 coat protein was incubated with DNase I and RNase A, but those in Lanes 1, 3, 5, 7, 9
approximately 14 kDa (Fig. 1c), and TEM results indicated and 11 were not. Lanes 1 and 2, MS2 VLPs; Lanes 3 and 4, GM-CSF
that these mRNAs were VLPs with diameters of about 30 nm VLPs; Lanes 5 and 6, mPAP VLPs; Lanes 7 and 8, hPAP VLPs; Lanes 9
and 10, mPAP-GM-CSF VLPs; Lanes 11 and 12, hPAP-GM-CSF VLPs;
(Fig. 1d). Lane M, molecular mass marker. (b) Purification of VLPs. The peak of
In prokaryotic expression systems, only 1,200 bases of a the target protein is marked by an arrow. (c) Verification of the pure
target RNA can be packaged with one wild type pac site;33 VLPs by SDS-PAGE. Lane 1, MS2 VLPs; Lane 2, GM-CSF VLPs; Lane 3,
therefore, we evaluated whether longer mRNAs could be mPAP VLPs; Lane 4, hPAP VLPs; Lane 5, mPAP-GM-CSF VLPs; Lane 6,
packaged in the presence of 1 C-variant pac site in a eukary- hPAP-GM-CSF; Lane M, molecular mass marker. (d) Further verification
of VLPs by TEM. (e) RT-PCR detection of mRNA packaged by the MS2
otic expression system. Our results indicate that the two capsid. Lanes 1, 2, 3, mPAP VLPs; Lanes 4, 5, 6, hPAP VLPs; Lanes 7,
tested longer RNA fragments (1,566 bp for mPAP-GM-CSF 8, 9, mPAP-GM-CSF VLPs; Lanes 10, 11, 12, hPAP-GM-CSF VLPs; Lanes
mRNA and 1,581 bp for hPAP-GM-CSF mRNA) could be 13, 14, 15, MS2 VLPs; Lanes 16, 17, 18, GM-CSF VLPs. Lanes 1, 4, 7,
successfully packaged by the MS2 capsid (Fig. 1e), and the 10, 13, 16, RT-PCR products of RNA extracted from related VLPs; Lanes
packaged sequence was further verified by sequencing. In 2, 5, 8, 11, 14, 17, negative controls (without RT); Lanes 3, 6, 9, 12,
15, 18, negative controls (corresponding to VLP sample). (f) Stability
addition, the nuclease-resistance assay showed MS2 VLP- assay of hPAP-GM-CSF VLPs. M, molecular mass marker; 1, no DNase I
based hPAP-GM-CSF mRNA was not degraded after it was or RNase A; 2–10, incubation with DNase I and RNase A for 2, 4, 6, 8,
incubated with DNase I (10 U/mL) and RNase A (10 mg/mL) 10, 12, 14, 16 or 18 hr, respectively. [Color figure can be viewed in the
at 37 C for up to 18 hr (Fig. 1f). These data reveal that MS2 online issue, which is available at wileyonlinelibrary.com.]
VLPs produced in yeast can specifically and completely pack-
age the 1,581-bp target mRNA with a C-variant pac site with MS2 VLP-based mRNA vaccines have strong translational
high yield, and because the MS2 VLP-based mRNA vaccines abilities in mammalian cells
are stable and bear the same capsid, they can be purified We investigated whether mRNA packaged by the MS2 capsid
using the same method. could be translated into protein in the mouse macrophage

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Figure 2. The translational and immune-stimulating ability of MS2 VLP-based mRNA. (a-c) Phagocytosis assay of the GFP VLPs in RAW264.7
cells. a, incubation with GFP VLPs unstained with FITC; b, incubation with GFP VLPs stained with FITC (under visible light); c, incubation with
GFP VLPs stained with FITC (under fluorescence). Original magnification, 2003. Images are representative of three independent experiments.
(d) The expression of MS2 VLP-based GFP mRNA in mouse macrophage cell line. (e) The clearance rates of GFP VLPs in mouse macrophages.
M, molecular mass marker; 1–5, incubation with MS2 VLPs; 6–10, incubation with GFP VLPs. 1 and 6, 120 hr; 2 and 7, 96 hr; 3 and 8, 72
hr; 4 and 9, 48 hr; 5 and 10, 24 hr. (f) Serum levels of IL-12 in C57BL/6 mice 4, 24 or 48 hr after intravenous injection with 15 lg, 50 lg or
200 lg of hPAP-GM-CSF VLPs or MS2 VLPs (n 5 5). **p < 0.01; ns, not significant; one-way ANOVA test. (g) Serum levels of TNF-a in C57BL/
6 mice 24 hr and 48 hr after intravenous injection with 15 lg, 50 lg or 200 lg of hPAP-GM-CSF VLPs or MS2 VLPs (n 5 5). **p < 0.01; ns,
not significant; one-way ANOVA test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

cell line, RAW264.7, with MS2 VLP-based GFP mRNA as the until RAW264.7 cells transformed and died. These results
model. A phagocytosis assay showed that, after 12 hr incuba- were different from those observed with the naked mRNA vac-
tion, a large number of FITC-labeled GFP VLPs were phago- cine, with which maximum expression emerged within 12–18
cytosed by RAW264.7 cells (Figs. 2a–2c). Next, the expression hr, with no detectable expression at 72 hr,10,34 proving the sta-
of GFP mRNA was monitored by fluorescence microscopy. bility of MS2 VLP-based mRNA vaccines.
After 48 hr incubation, faint green fluorescence was observed The clearance rates of MS2 VLP-based GFP mRNA were
in almost all RAW264.7 cells. As the incubation time pro- detected by RT-PCR. GFP mRNA in RAW264.7 cells
gressed, the intensity of green fluorescence was enhanced, with increased within the first 4 days and decreased sharply on
the peak appearing at 72 hr (Fig. 2d), and it was maintained Day 5 (Fig. 2e), even though GFP VLPs were still present in

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the supernatant on Day 5. This implies a limited and dose- T cell proliferation was detectable in the mPAP group, mPAP-
dependent phagocytosis ability of macrophage cell line. To GM-CSF group, hPAP group, and hPAP-GM-CSF group but
validate this conclusion, different numbers (103, 104, 105 and not in the MS2 group and GM-CSF group. The amount of
106) of cells were co-cultured with 10 lg/mL GFP VLPs. We PAP-specific CD41 and CD81 T cells in hPAP-GM-CSF mice
found that 103 cells transformed completely after incubation was significantly different from that of hPAP mice and mPAP-
with GFP VLPs for 24 hr, and 104 cells did so after 48 hr, GM-CSF mice, respectively (Figs. 3c and 3d). Furthermore, for
but the majority of cells did not completely transform until the mPAP-GM-CSF group, mPAP-specific CD41 T cell prolif-
120 hr. These results suggest that the mRNA packaged by eration was higher than that for the mPAP group, as was that of
the MS2 capsid can be translated into protein in mammalian mPAP-specific CD81 T cells. All these results suggest that MS2
cells, and the translation is dose and time dependent. VLP-based mRNA vaccines elicit strong humoral and cellular
immune responses, including CD41 T cell and CTL responses,
Immune-stimulating activity of MS2 VLP-based mRNA the former being potent mediators of anti-tumor immunity35
In order to determine whether MS2 VLP-based hPAP-GM- and associated with concomitant memory response, and the lat-
CSF mRNA can be used as a vaccine, the immune-stimulating ter playing an important role in the immune defense against
activity of this MS2 VLP-based mRNA was evaluated in cancer.36 Moreover, xenogeneic mRNA induces stronger
C57BL/6 mice. Mice were immunized intravenously with immune responses than syngeneic mRNA, which is similar to
hPAP-GM-CSF VLPs, and the levels of serum IL-12 and TNF- protein3 and DNA37 vaccines, and murine GM-CSF mRNA
a were analyzed by ELISA. Compared to MS2 VLPs and PBS, appears to play an important adjuvanticity in augmenting these
hPAP-GM-CSF VLPs caused up-regulation of IL-12 and TNF- responses. From the T cell proliferation assay, we further con-
a, and the immune-stimulating activity of 50 lg and 200 lg cluded that the protein translated from the packaged mRNA
per mouse was stronger than that of 15 lg per mouse (Figs. 2f may play a key role in priming the T cell response.
and 2g), indicating MS2 VLP-based hPAP-GM-CSF mRNA We next detected the frequencies of Treg cells in peripheral
had potent immune-stimulating activity in vivo, which was blood from vaccinated and control mice (Fig. 3d and Supporting
associated with the inoculation amount. Information Fig. S4). We found that neither mPAP-GM-CSF
mRNA nor hPAP-GM-CSF mRNA changed the frequencies of
MS2 VLP-based mRNA vaccines induce strong humoral and Treg cells, compared to the frequencies of Treg cells measured in
cellular immune responses other groups, including a normal group (control). Moreover,
We explored whether MS2 VLP-based mRNA vaccines could intensive vaccination did not increase the frequencies of Treg
Tumor Immunology

elicit antigen-specific immune responses in C57BL/6 mice. cells. This means that the MS2 VLP-based mRNA vaccines used
First, sera antibodies were detected following prophylactic here did not alter the immune homeostasis of mice.
vaccination. PAP-specific antibodies were elicited by immuni-
zation with mPAP VLPs, hPAP VLPs, mPAP-GM-CSF VLPs MS2 VLP-based mRNA vaccines trigger balanced Th1/Th2
and hPAP-GM-CSF VLPs but not with MS2 VLPs and GM- responses
CSF VLPs (Fig. 3a). Of note, the antibody titers in the Because antigen-specific CD41 T cells were induced by
mPAP-GM-CSF and hPAP-GM-CSF groups were signifi- immunization with MS2 VLP-based mRNA vaccines, we
cantly higher than those in the mPAP and hPAP groups, wanted to know which type of Th-like immune response was
respectively. However, there was no significant difference in elicited. Both subtypes of PAP-specific IgG antibodies in
antibody levels between the mPAP-GM-CSF group and the serum were detected 14 days after the last vaccination by
hPAP-GM-CSF group. After boosted vaccinations, the IgG ELISA. PAP-specific IgG1 and IgG2a subtype antibodies were
titers became elevated, and then dropped slightly 30 days significantly elicited in the mPAP, mPAP-GM-CSF, hPAP
after the last immunization but were not significantly differ- and hPAP-GM-CSF groups compared to the other control
ent from those of 14 days. groups (MS2, GM-CSF and PBS; DOD450 < 0.050) (Fig. 4a).
Next, to assess the ability of MS2 VLP-based mRNA vac- Compared with the PAP groups (mPAP and hPAP), the
cines to induce an antigen-specific T-cell immune response, titers of PAP-specific IgG1 and IgG2a were enhanced signifi-
we separated splenocytes from vaccinated and control mice. cantly (p < 0.001), and the ratios of IgG1/IgG2a increased
After stimulation with PAP protein, splenocytes from mice (mPAP vs. mPAP-GM-CSF: 5.21 6 0.98 vs. 2.85 6 0.90, p 5
vaccinated with four VLPs (mPAP VLPs, mPAP-GM-CSF 0.0017; hPAP vs. hPAP-GM-CSF: 4.36 6 1.91 vs. 2.36 6
VLPs, hPAP VLPs and hPAP-GM-CSF VLPs) secreted IFN-g, 0.65, p 5 0.0089) by the addition of the adjuvant, GM-CSF.
but splenocytes from mice vaccinated with MS2 VLPs or GM- In addition, IgG1 titers in the mPAP-GM-CSF group were
CSF VLPs did not (Supporting Information Fig. S3). The markedly higher than those in the hPAP-GM-CSF group, but
amounts of secreted IFN-g in hPAP-GM-CSF and mPAP- IgG2a was not, suggesting that the hPAP-GM-CSF mRNA
GM-CSF groups were significantly different (p 5 0.0079), and vaccine might have more potential in inducing balanced Th1
were higher than those in hPAP and mPAP groups. and Th2 immune responses.
The results of T cell proliferation assay indicated that after The Th1-biased immune response was confirmed by cyto-
the stimulation of PAP protein, PAP-specific CD41 and CD81 kine detection (Figs. 4b–4e). IFN-g, TNF-a, IL-12, IL-6 and

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Figure 3. Antigen-specific antibody and cellular immune response induced by MS2 VLP-based mRNA vaccines. The graphs show the mean 6
SEM of these data for all animals per treatment group. (a) Measurement of PAP-specific IgG antibody levels in serum before and after the
boosted immunizations by ELISA (n 5 6–10). Arrows indicate vaccination time (Two-way ANOVA test and Bonferroni post test). (b) PAP-specific
IgG antibody titers on Day 14 after the third immunization (one-way ANOVA test). (c, d) Evaluation of PAP-specific T cell proliferation in spleno-
cytes (n 5 5) by FACS (one-way ANOVA test). (e) Frequencies of CD41CD251Foxp31 T cells within CD41 T cells in peripheral blood isolated from
vaccinated and control mice on Day 7 after the last vaccination (n 5 5) (one-way ANOVA test). ns, not significant; **p < 0.01; ***p < 0.001.
[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

IL-4 in the culture supernatant from stimulated splenocytes GM-CSF) groups (Fig. 4d). Notably, there were no significant
were detected by ELISA. Compared to the mPAP group, differences in the amounts of IL-6 in above two groups (Fig.
mPAP-GM-CSF group and hPAP group, the splenocytes of 4e). However, after PAP stimulation, the amounts of IL-4
mice from the hPAP-GM-CSF group released more PAP- were below the detection limit in the culture supernatants of
specific IFN-g and IL-12 (Figs. 4b and 4c). In addition, the T cells derived from all mice. In contrast, after stimulation
concentration of TNF-a in the mPAP-GM-CSF and hPAP- with MS2 VLPs, IL-4 and IL-6 were found in higher levels
GM-CSF groups was higher than in the groups without GM- (Supporting Information Fig. S5). These data indicate that
CSF, but there was no apparent difference between mPAP the MS2 coat protein induces a Th2-like immune response,
(with and without GM-CSF) and hPAP (with and without but this response is converted to a Th1-biased response by

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Figure 4. Balanced Th1/Th2 immune response elicited by MS2 VLP-based mRNA vaccines. The data show the mean 6 SEM of DOD450
obtained from five mice per treatment group. (a) Detection of serum IgG1 and IgG2a antibodies from vaccinated and control mice by ELISA.
Sera were collected on Day 14 after the last immunization. (b–e) Levels of cytokines (IFN-g, TNF-a, IL-12 and IL-6) in culture supernatant
after PAP protein stimulation. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant; one-way ANOVA test.

the target protein translated from the packaged mRNA, polarization of immunity from Th2 to Th1, we wanted to
which is further enhanced by GM-CSF. identify the potential mechanisms underlying these responses.
To address this question, we assessed whether mouse DCs
Activation of mouse DCs in vivo by MS2 VLP-based mRNA were activated in vivo after vaccination with MS2 VLP-based
vaccines mRNA vaccines. This was confirmed by FACS on Day 7 after
After verifying that MS2 VLP-based mRNA vaccines stimu- inoculation of the MS2 VLP-based mRNA vaccines (Fig. 5a).
lated humoral and cellular immune responses and induced As expected, the expressions of mouse DC surface markers

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Figure 5. Prophylactic immunization with MS2 VLP-based hPAP-GM-CSF mRNA vaccine protects C57BL/6 mice against PCa. (a) FACS analysis
of mean fluorescent intensity (MFI) of CD11c1 cells expressing maturation markers in different groups (n 5 5). *p < 0.05; **p < 0.01; one-
way ANOVA test. (b) The size of tumors separated on Day 20 after challenge (n 5 5). (c) Mean survival following the challenge of TRAMP-
C2 cells (n 5 10). hPAP-GM-CSF group versus hPAP group, p < 0.05; log-rank test. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]

CD11c, CD40, CD83, CD86 and MHC II were drastically up- were subcutaneously challenged on Day 42 with 1 3 106
regulated in the groups co-immunized with GM-CSF mRNA TRAMP-C2 PCa cells (Figs. 5b and 5c and Supporting Infor-
(mPAP-GM-CSF group and hPAP-GM-CSF group), com- mation Fig. S6). Compared with MS2 VLPs and PBS, pro-
pared to the other groups. Furthermore, the expression levels phylactic immunization with hPAP-GM-CSF, hPAP, mPAP-
of CD40 and CD86 in the hPAP-GM-CSF group were higher GM-CSF, mPAP or GM-CSF VLPs delayed tumor growth for
than those in the mPAP-GM-CSF group (p < 0.05). This 1–3 weeks (Supporting Information Fig. S6). On Day 20, no
suggests that when target mRNA and GM-CSF mRNA are tumors were found in the mice belonging to the hPAP-GM-
administrated together at a 1:1 molar ratio, GM-CSF mRNA CSF group (Fig. 5b), and the mice in this group survived
triggers the maturation of DCs, and the mature DCs then completely (10/10) (Fig. 5c) until the observation period
further prime B cell and T cell immunity by presenting anti- ended (60 days after tumor cell challenge), whereas all mice
gen from the translation of the packaged mRNA. Impor- in the other groups succumbed to tumor challenge. This
tantly, as an adjuvant, GM-CSF mRNA is easier to prepare observation supports the ideas that MS2 VLP-based hPAP-
and use than its protein27 and DNA.38 GM-CSF mRNA vaccines are able to suppress prostatic
tumor growth in C57BL/6 mice and that the xenogeneic
Prophylactic and therapeutic effect of MS2 VLP-based mRNA vaccine has a more potent antitumor effect than the
hPAP-GM-CSF mRNA vaccines syngeneic mRNA vaccine, as xenogeneic protein and DNA
We evaluated the antitumor activity of MS2 VLP-based vaccine have been proven effective.26,39,40
hPAP-GM-CSF mRNA nanoparticles in mouse models of Considering that hPAP-GM-CSF VLPs protected mice
PCa. C57BL/6 mice were vaccinated intravenously with the 6 against tumor challenge prophylactically, we also investigated
purified VLPs mentioned above on Days 0, 14 and 28 and whether they can control tumor growth under therapeutic

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 1692 The packaged RNA vaccine for prostate cancer

and special laboratories. Hence, RNA delivery in vivo, which

does not need complicate procedures and can directly target
antigen-presenting cells, would be preferable. Direct injection
of naked synthetic mRNA has proven to be feasible and safe
and induces antigen-specific immune responses in a clinical
setting.41 However, the instability of mRNA influences the
efficacy of an mRNA vaccine. To develop an mRNA vaccine
that is resistant to rapid extracellular degradation, this study
adopted an MS2 VLP-based RNA approach, in which, the
target mRNA is packaged by the coat protein of the MS2
bacteriophage and not degraded within 18 hr, which is
dependent on the highly specific interaction between the coat
protein and pac site.42 In our study, MS2 VLP-based mRNAs
were expressed in S. cerevisiae with high-efficiency and were
purified easily; they were also nuclease-resistant, non-replica-
tive and non-infectious. Furthermore, no other physical evi-
dence of toxicity was observed, except for bruising at the
injection sites, which showed that these mRNA vaccines were
safe.
Exogenous mRNA packaged by the MS2 capsid is success-
fully translated into bio-active whole protein in eukaryotes
and has stronger immunogenicity than peptide-based vac-
cines. Moreover, the MS2 VLP-based PAP mRNA vaccines
induced the expression of IL-12 and other cytokines, indicat-
ing the activation of the innate immune system.
Xenogeneic protein or DNA vaccination induces a more
potent immune response than syngeneic material.26,39,40 Our
results demonstrated that immunization with MS2 VLP-
Tumor Immunology

based xenogeneic and syngeneic mRNA vaccines elicited

Figure 6. MS2 VLP-based hPAP-GM-CSF mRNA vaccine induces anti-
humoral, balanced Th1/Th2 PAP-specific CD41, and CD81
tumor response under therapeutic conditions. (a) Time course of
mean tumor growth. Tumor sizes of the mice were measured in T cell responses. However, even though immunization with
three dimensions using calipers once a week and within a group syngeneic mRNA vaccine overcame self-tolerance against
were calculated as the arithmetic mean of the tumor volumes with mPAP and produced a little higher titer of antibody, a more
SEM. **p < 0.01; ns, p > 0.05; two-way ANOVA test and Bonfer- potential anti-tumor effect was observed in mice vaccinated
roni post test. (b) Survival time and survival rates after tumor chal-
with MS2 VLP-based xenogeneic mRNA, which may be
lenge. hPAP-GM-CSF group versus hPAP group, p < 0.05; log-rank
test. [Color figure can be viewed in the online issue, which is avail- mediated by Th1 and CTL immune responses and not by a
able at wileyonlinelibrary.com.] humoral immune response. In other words, a stable antitu-
mor effect may be not highly correlated with the strong
conditions. The treatment of tumor with hPAP-GM-CSF humoral immune response, but correlated with strong cellu-
VLPs, hPAP VLPs or mPAP-GM-CSF VLPs led to a tumor lar immune response, especially CTL response, which may
growth delay of about one week, whereas treatment of tumor explain why the inability of mPAP-GM-CSF to provide pro-
with the other VLPs was not effective (Fig. 6a). Also, hPAP- tection. To our knowledge, this is the fist report of MS2 VLP
GM-CSF VLPs inhibited tumor growth significantly than expressing a xenoantigen and choosing xenogeneic mRNA
hPAP and mPAP-GM-CSF VLPs. Subsequently, survival time for cancer vaccination. Therefore, this study provides new
and survival rates were evaluated (Fig. 6b). Treatment with thinking for the development of PCa immunotherapy.
hPAP-GM-CSF VLPs prolonged at least 10 days compared The adjuvant properties of the GM-CSF mRNA were also
with the other VLPs, but did not improve the survival rate. demonstrated in this study. Our data suggested, when the
This indicates the immune responses induced by hPAP-GM- molar ratio of PAP to GM-CSF mRNA was 1:1, GM-CSF
CSF VLPs are not strong enough to fight against the rapid mRNA augmented humoral and cellular immune responses
growth of tumor. induced by an MS2 VLP-based PAP mRNA vaccine and
boosted Th1-like immune response by promoting the matu-
Discussion ration of DCs. These results are identical to those of a previ-
Most studies on mRNA vaccine have been limited to loading ous study.29
DCs with RNA in vitro, which is not only time-consuming In the study by Johnson et al.,43 PAP DNA vaccine could
and cost-intensive but also needs professional technologists not induce PAP-specific antibody and T cell immune

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response without the adjuvant GM-CSF, but in this study, control; (ii) shows high yield in a yeast expression system;
PAP mRNA vaccine could. Moreover, these responses (iii) has higher stability (for at least 18 hr) when incubated
induced by PAP mRNA were stronger than those induced by with a nuclease; (iv) is suitable for phagocytosis and proper
PAP DNA. protein expression, which can overcome self-tolerance, acti-
As a prophylactic vaccine, the MS2 VLP-based hPAP-GM- vate DCs and induce efficient immune responses; (v) is safe,
CSF mRNA vaccine completely protected mice from PCa, without obvious adverse effects when administrated intrave-
which proved that this vaccine had better preventive effect than nously; (vi) simultaneously induces humoral and cellular
liposome-encapsulated mRNA vaccine44,45 and the same effect immune responses; (vii) generates CD41 T helper cells, espe-
as naked mRNA vaccine.46 As a therapeutic vaccine, MS2 VLP- cially a Th1-biased response, which is required for the pro-
based hPAP-GM-CSF mRNA vaccine also inhibited the growth duction of effector and memory CD81 T cells; (viii)
of established tumors, implying this vaccine could evoke an effi- maintains a balance between CD41 T cells and Treg cells;
cient immune response in a short time, however, this response (ix) produces a CTL response; (x) shows strong tumor inhibi-
is not sufficient to overcome the tumor. Moreover, MS2 VLP- tion effects; and (xi) MS2 capsid can be modified by inserting
based hPAP mRNA vaccine was inferior to hPAP-GM-CSF peptides, which make it targeted for specific cells. Compared
mRNA vaccine, further demonstrating GM-CSF had important with published VLP-based peptide vaccines,48–50 MS2 VLP-
adjuvanticity in activating the immune system. based vaccine can be used as mRNA-peptide combination
In a previous study, Treg cells controlled key aspects of vaccines, due to the abilities of packaging specific exogenous
immunologic tolerance.47 However, we found that repeated mRNA and displaying peptides of MS2 VLP.18 Hence, MS2
vaccination with MS2 VLP-based mRNA vaccines did not VLP-based mRNA vaccine strategy is an exciting proposition
induce the upregulation of Treg cells. We speculate that the for the development of mRNA vaccines as well as mRNA-
limited antigen expression level and time after vaccination peptide combination vaccines.
support a more favorable balance between CD41 T cells and
Treg cells. Acknowledgements
In conclusion, MS2 VLP-based mRNA vaccine strategy we The authors thank Mark J Zylka in University of North Car-
have discussed here has the following characteristics, which olina, USA and Jiyun Yu in the Academy of Military Medical
make it a promising cancer vaccine: (i) can be prepared easily Sciences, China for providing mouse PAP and GM-CSF
by recombinant protein technology, and the quality is easy to cDNAs, respectively.

Tumor Immunology
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